Hepatic steatosis, also known as non-alcoholic fatty liver disease (NAFLD), is an increasingly common pediatric and adult condition that is associated with central adiposity, dyslipidemia, insulin resistance, and type 2 diabetes. While initially a reversible condition without significant clinical symptomatology, NAFLD progresses in 15-20% of patients to non-alcoholic steatohepatitis (NASH), an inflammatory condition that can lead to cirrhosis and hepatic failure. Epidemiological trends showing higher rates in males than females until menopause suggest that estrogen may protect against the development of NAFLD. This concept is also supported by animal research showing increased rates of hepatic steatosis in estrogen-deficient aromatase knockout mice; furthermore, estrogen receptor knockout mice (ER1 KO) exhibit other metabolic features associated with NAFLD, including increased weight, altered body fat distribution, and insulin resistance. Preliminary findings from this laboratory indicate that both ER-1 KO male and female mice develop hepatic steatosis and insulin resistance in response to high-fat feeding. Therefore, we propose to employ the ER1 KO model to clarify the mechanisms by which estrogen protects against hepatic fat deposition. To that end, we propose two Specific Aims. First, we will generate a liver-specific ER1 knockout mouse by breeding global ER1 KO mice with mice expressing cre-albumin; the resultant liver-specific knockout will be compared to whole body ER1 KO mice to determine if the effects of estrogen on liver fat storage are mediated locally or linked to more peripheral actions of estrogen signaling in adipocytes or muscle. Second, we will compare the hepatic and metabolic phenotypes of ER1 KO mice with those of mice engineered to express ER1 lacking the capacity for direct DNA binding. The latter respond to estrogen through tethering interactions with target gene sequences. These studies will elucidate the basic molecular mechanisms by which ER1 signaling prevents excess liver fat deposition. Understanding the molecular mechanisms of estrogen action is essential for the development of pharmacotherapies targeted to protect against metabolic dysfunction; our studies could prove invaluable in designing future treatments to protect those at risk for the development or progression of NAFLD.